Fig. 3.

Effects of GRHL2 overexpression in combination with DNA methyltransferase (DNMT) inhibitor (5-azacitidine (5-aza)) treatment. a Western blots of ZEB1, E-cadherin, GRHL2, vimentin, and β-actin in OVCA429 shGRHL2 Tet-GRHL2* cells with/without doxycycline (Dox)-induced GRHL2 overexpression for 48 and 96 h, along with/without 5-azacitidine treatment. Representative blots from three independent experiments are shown. Bar graphs (bottom) showing messenger RNA (mRNA) fold changes of GRHL2, CDH1, VIM, ZEB1 in OVCA429 shGRHL2 Tet-GRHL2* cells with/without 96 h doxycycline-induced GRHL2 overexpression, along with/without 5-aza treatment. Error bars = s.e.m. Unpaired t tests were used on independent triplicates, *0.01 < p < 0.05; **p < 0.01. b Same as in a but in the HEYA8 Tet-GRHL2 model. c Images of immunofluorescence staining showing GRHL2 (green) and E-cadherin (red) after GRHL2 knockdown or GRHL2 overexpression in the intermediate epithelial–mesenchymal transition (EMT) cell lines OVCA429 and IOSE523 and in the full EMT cell line HEYA8. Nuclei were stained blue (DAPI (4′,6-diamidino-2-phenylindole)). Scale = 50 μm. d Dot plots showing the mean mRNA fold changes (red lines) of CLDN4, PROM2, CGN, PVRL4, S100A14, SPINT1 in OVCA429 shGRHL2 Tet-GRHL2* (left) and HEYA8 Tet-GRHL2 (right) with/without 96 h doxycycline-induced GRHL2 overexpression, along with/without 5-aza treatment (data from independent triplicates). Error bars = s.e.m. Unpaired t tests were performed: *0.01 < p < 0.05; **p < 0.01. KD, knock down